The broad goal of this proposal is to obtain high- resolution structural information on membrane-bound annexins (ANXs) that is critical for understanding the biophysical mechanism by which these proteins interact with bilayers and for evaluating hypotheses concerning their biological functions. ANXs are a superfamily of proteins with structurally conserved core domains that mediate reversible binding to phospholipid membranes and thereby elicit a number of intriguing effects due to perturbation of bilayer structure; e.g. many ANXs exhibit ion channel activity. Each ANX gene product also has a short structurally unrelated N-terminal domain that has a unique function; e.g. the N-terminal domain of ANXA2 binds and stimulates tissue plasminogen activator (t-PA). The proposed studies will focus on two ANXs: ANXB12 because previous studies establish it as archetype of core domain structure and ANXA2 because it offers attractive opportunities to connect structure to biological function. Published studies ANXB12 showed that it exists in three structural forms: a soluble monomer, a Ca2+- dependent peripheral membrane-bound trimer, and a Ca2+- independent transmembrane channel that is the result of a dramatic "inside-out" refolding of the core domain. These three forms undergo reversible interconversion with the equilibrium being modulated by phospholipid, Ca2+ and H+. Biochemical, site- directed spin labeling and other experimental approaches will be used to determine the structures of the membrane-bound forms of ANXB12 and ANXA2 and evaluate our hypothesis that protonation of carboxylate switch residues regulates the equilibrium. The "carboxylate switch" structural motif proposed to mediate the transmembrane insertion of ANXs is found in a variety of biologically important proteins and may define a novel class of membrane proteins. In addition, the structure of the N-terminal domain of ANXA2 will be investigated in intact endothelial cells with the goal of determining the molecular mechanism by which ANXA2 activates t-PA. Characterization of the ANXA2 binding site for t-PA is the first step toward the design of drugs that modulate the ANXA2-dependent fibrinolysis pathway.